VETERINARY SERVICES MEMORANDUM NO. 800.102

December 12, 2013

TO: Veterinary Services Leadership Team

Directors, Center for Veterinary Biologics

Biologics Licensees, Permittees, and Applicants

FROM: John R. Clifford /s/ Jack A. Shere, for

Deputy Administrator

SUBJECT: Exemption from Leptospira Bacterin Testing Under 9 CFR 

113.101-104 and the Associated References and Studies

I. PURPOSE

This memorandum provides guidance for obtaining an exemption to the 

requirement for testing Leptospira bacterins for potency in hamsters. It also 

provides information on alternative in vitro potency assays and reagents available 

from the Center for Veterinary Biologics (CVB).

II. REPLACEMENT

This memorandum replaces Veterinary Services (VS) Memorandum No. 800.102, 

dated May 23, 2002. In addition, it compiles information on policies and reagents

supplied in the following CVB notices:

1. CVB Notice No. 07-02, “Qualification of Leptospira grippotyphosa and 

Leptospira icterohaemorrhagiae Reference Bacterins for Products Intended for 

Use in Dogs,” dated March 1, 2007. 

2. CVB Notice No. 07-12, “Qualification of Leptospira pomona and Leptospira 

canicola Reference Bacterins for Products Intended for Use in Dogs,” dated 

July 13, 2007.

3. CVB Notice No. 09-16, “Qualification of Leptospira canicola, Leptospira 

grippotyphosa, Leptospira icterohaemorrhagiae, and Leptospira pomona

Reference Bacterins for Products Intended for Use in Swine and/or Cattle,” dated 

August 3, 2009.

III. BACKGROUND

Of the several animal models established for leptospirosis, hamsters are 

commonly used due to their high susceptibility to infection. The hamster model 

is used in Leptospira bacterin potency tests codified as Standard Requirements in 

title 9, Code of Federal Regulations (9 CFR), parts 113.101-104. The codified 

Animal and Plant

Health Inspection

Service

Veterinary Services

1400 Independence 

Ave, SW

Washington, DC 

20250
VETERINARY SERVICES MEMORANDUM NO. 800.102

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tests utilize a vaccination challenge format in a hamster model. Product is diluted based 

on the host animal dose volume. The route used is described as “in accordance with label 

recommendations for use.”

The CVB considers exemptions to codified tests, per 9 CFR 113.4 (a), if the replacement 

test is at least equivalent to the Standard Requirement and the test methods are described 

in the filed Outline of Production for the product.

In keeping with its commitment to replace animal tests with in vitro tests, enzyme-linked 

immunoassays (ELISA) and associated reagents for potency testing Leptospira products 

have been developed by the CVB. Test details are provided in Supplemental Assay 

Methods (SAM) 624-627, and reagents are available per VS Memorandum No. 800.97.

We encourage firms to obtain exemptions to use the ELISA tests.

The ELISAs require a suitably qualified reference bacterin. The CVB supplies nonadjuvanted Leptospira reference bacterins that may be used to test products intended for 

use in dogs, cattle, or swine. The stability of the Standard Reference Bacterins will be 

monitored by the CVB via performance in the relevant codified hamster test. New 

Standard Reference Bacterins will be qualified using the relevant codified hamster test.

The availability of Standard Reference Bacterins does not preclude the use of a suitable 

qualified alternative reference developed by the licensee, and an alternative reference 

may be necessary for certain products. Qualifying a new reference bacterin typically 

involves the demonstration that the reference is acceptably efficacious in a host animal 

vaccination-challenge model. A surrogate animal model may be used in lieu of host 

animal studies to qualify a reference (not for pivotal efficacy), provided that the surrogate 

animal model is deemed to be an acceptable indicator of efficacy in the host animal. The 

CVB conducted studies to confirm that the codified hamster test is a suitable surrogate to 

evaluate the potency of the following Leptospira fractions in dogs, cattle, and swine: 

Leptospira serogroups canicola, grippotyphosa, icterohaemorrhagiae, and pomona.

IV. POLICY

A. Host Animal Efficacy Studies. Although the codified hamster test is considered 

to be correlated to host animal efficacy, in most cases it does not reflect the 

natural transmission of the pathogen in the host animal. Therefore, the CVB 

requires host animal efficacy studies to license products containing new Master 

Seed Bacterins (MSB) or to evaluate substantial production method changes. The 

antigen input of the efficacy serial should be characterized in objective terms 

(e.g., organism counts prior to inactivation), not as potency relative to a reference, 

and the efficacy serial should also pass the hamster test. If these conditions are 

met, subsequent reference bacterins prepared to contain at least as much antigen 

as the efficacy serial may be qualified using the relevant codified hamster test.

Summarize host animal efficacy studies in section V.C of the Outline of 

Production, and include the challenge strain identified to the serogroup.

 
VETERINARY SERVICES MEMORANDUM NO. 800.102

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B. Codified Hamster Test. The route used in the hamster potency test should be 

consistent with the label recommendations for the host animal.

Host Animal routes Codified Hamster Test routes

Efficacy study Labeling

Hamster 

vaccination route 

for Serial release

Hamster 

Vaccination route 

for ELISA reference 

maintenance

IM IM IM IM

SC SC SC SC

IM & SC IM & SC IM or SC but must 

specify in Outline

IM or SC but must 

specify in Outline

IM & SC SC SC SC

IM & SC IM IM IM

When the hamster test is used, we encourage firms to use humane end 

points wherever possible.

C. Exemptions to use ELISA based tests. The licensee or permittee must request an 

exemption to tests prescribed in 9 CFR 113.101-104. The ELISA procedure must 

be described in the relevant Outlines. To maintain the exemption, the minimum 

antigen content specified in section IV of the Outline must not be reduced from 

that established at licensing. Prelicense products must meet all current 

requirements for establishment of minimum antigen content. Cite the exemption 

as described in VS Memorandum No. 800.206.

Submit the following supporting information to request an exemption to codified 

potency testing:

1. MSB. Identify the MSB to the serogroup level to ensure the reagents utilized 

in the ELISA protocol are appropriate.

2. Production methods should be detailed to ensure significant changes can be 

noted.

3. Validate the performance of the ELISA with the specific product for which 

the exemption is sought. Follow the guidance in VS Memorandum No. 

800.112 to generate specificity, reproducibility, and parallelism data. 

Leptospira bacterins do not react identically in the ELISA; firms with some 

data collected to validate the test may submit preliminary data for CVB 

review and comment.

 
VETERINARY SERVICES MEMORANDUM NO. 800.102

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D. Identification of a reference bacterin. The need for a qualified reference may be 

addressed by one of the following methods, provided the potency assay can be 

acceptably validated using the proposed reference, as indicated above.

1. Standard Reference Bacterin. Use the Standard Reference Bacterin supplied 

by the CVB at the recommended working dilution. The Standard Reference 

Bacterin can be used to release product labeled as an aid in the prevention of 

disease due to the applicable Leptospira in dogs, cattle, or swine.

2. Product-matched Reference Bacterin (qualified by hamster test). Produce a 

product-matched reference bacterin containing at least as much antigen input 

as the efficacy serial and qualify it via the codified hamster potency test.

Submit data from at least three independent codified tests (beginning with 

three separate vials of product). If the reference will be used to test products 

that are recommended for subcutaneous injection, vaccinate the hamsters 

subcutaneously. Suitable references should repeatedly generate satisfactory 

results in the codified test. All candidate reference preparations will be 

subject to confirmatory testing by 9 CFR 113.101-104, as appropriate, before 

final Animal and Plant Health Inspection Service approval is granted.

This approach may be required for adjuvanted products that do not 

demonstrate acceptable parallelism with the non-adjuvanted Standard 

Reference Bacterin in the potency assay. References qualified in this manner 

can be used to release product labeled as an aid in the prevention of disease 

due to the relevant Leptospira in the relevant host animal.

3. Host qualified reference bacterin. If data submitted to support the hamster 

test is not appropriate for a product, this may be considered by the CVB on a 

case-by-case basis. For those references which are not suitable for 

monitoring in hamster assay, the reference should be requalified in the host 

animals at regular intervals of five years. 

E. Valid Challenge Ranges. When the hamster vaccination challenge tests are 

conducted via 9 CFR 113.101-104, a range of 10-10,000 hamster LD50 is 

codified. Valid test results are not obtained at challenge doses above 10,000 

LD50. This may be due to an immunizing effect or the induction of a chronic 

disease state. Over-challenges are considered a no test and must be repeated. No 

changes to this policy will be considered unless additional guidance is released.

F. Stability Monitoring of References

1. Standard Reference Bacterins will be monitored by CVB utilizing the 

hamster test.

 
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2. Firm’s reference bacterins should be firm monitored using trend analysis and 

periodic reevaluation (approximately every 2.5 years) using the codified 

hamster test. The CVB may monitor the firm’s reference bacterins at their 

discretion via the codified hamster potency assay. For products unable to be 

monitored by the hamster laboratory model, please refer to section IV.D.3.

V. IMPLEMENTATION/APPLICABILITY

This memo is applicable immediately and applies to all Leptospira bacterins with 

codified potency tests and intended for use in dogs, cattle, or swine. It applies to serials 

manufactured after the signature date of this memo. Firms not in compliance with this 

memorandum should contact their assigned reviewer to propose alternative strategies.